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Ambrosia beetles are highly successful as invaders because they are often transported internationally with wood packaging and other wood products and because their inbreeding mating systems facilitates establishment of invading populations. In 2022, two independent insect surveys in canton Ticino (southern Switzerland) revealed the widespread occurrence of the invasive ambrosia beetle Anisandrus maiche (Kurentzov, 1941) from southern to central-upper Ticino. This species is native to east Asia and has previously been found as a non-native invasive species in the United States, Canada, western Russia, Ukraine and, in 2021, in northern Italy. Here, we present the results of several trapping studies using different trap types (bottle traps, funnel traps and Polytrap intercept traps) and attractants and a map of the distribution of the species. In total, 715 specimens of A. maiche, all female, were trapped, and the identity of selected individuals was confirmed by morphological and molecular identification based on three mitochondrial and nuclear markers (COI, 28S and CAD). Trap samples from early April to early September 2022 in intervals of two to four weeks showed that flights of A. maiche occurred mainly from June to mid-August. Isolation of fungal associates of A. maiche from beetles trapped alive revealed the presence of four fungal species, including the ambrosia fungus Ambrosiella cleistominuta, the known mutualist of A. maiche. The identity of A. cleistominuta was confirmed by comparing DNA sequences of its nuclear, internal transcribed spacer (ITS) gene with reference sequences in NCBI and BOLDSYSTEMS. This represents the first record of A. cleistominuta in Europe. Of the other fungal associates isolated from A. maiche in Ticino, Fusarium lateritium is of note as there is a possibility that A. maiche could act as a vector of this plant pathogen. We highlight several research needs that should be addressed to gain insight into the potential impact of these non-native species and to overcome problems with heteroplasmy in COI sequences in studies of invasion and population genetics of ambrosia beetles.

ABSTRACTWe previously demonstrated that mutation of sarA in Staphylococcus aureus limits biofilm formation, cytotoxicity for osteoblasts and osteoclasts, and virulence in osteomyelitis, and that all of these phenotypes can be attributed to the increased production of extracellular proteases. Here we extend these studies to assess the individual importance of these proteases alone and in combination with each other using the methicillin-resistant USA300 strain LAC, the methicillin-susceptible USA200 strain UAMS-1, and isogenic sarA mutants that were also unable to produce aureolysin (Aur), staphopain A (ScpA), staphylococcal serine protease A (subsp.), staphopain B (SspB), and the staphylococcal serine protease-like proteins A-F (SplA-F). Biofilm formation was restored in LAC and UAMS-1 sarA mutants by subsequent mutation of aur and scpA, while mutation of aur had the greatest impact on cytotoxicity to mammalian cells, particularly with conditioned medium (CM) from the more cytotoxic strain LAC. However, SDS-PAGE and western blot analysis of CM confirmed that mutation of sspAB was also required to mimic the phenotype of sarA mutants unable to produce any extracellular proteases. Nevertheless, in a murine model of post-traumatic osteomyelitis, mutation of aur and scpA had the greatest impact on restoring the virulence of LAC and UAMS-1 sarA mutants, with concurrent mutation of sspAB and the spl operon having relatively little effect. These results demonstrate that the increased production of Aur and ScpA in combination with each other is a primary determinant of the reduced virulence of S. aureus sarA mutants in diverse clinical isolates including both methicillin-resistant and methicillin-susceptible strains.IMPORTANCEPrevious work established that SarA plays a primary role in limiting the production of extracellular proteases to prevent them from limiting the abundance of S. aureus virulence factors. Eliminating the production of all 10 extracellular proteases in the methicillin-resistant strain LAC has also been shown to enhance virulence in a murine sepsis model, and this has been attributed to the specific proteases Aur and ScpA. The importance of this work lies in our demonstration that the increased production of these same proteases largely accounts for the decreased virulence of sarA mutants in a murine model of post-traumatic osteomyelitis not only in LAC but also in the methicillin-susceptible human osteomyelitis isolate UAMS-1. This confirms that sarA-mediated repression of Aur and ScpA production plays a critical role in the posttranslational regulation of S. aureus virulence factors in diverse clinical isolates and diverse forms of S. aureus infection.

ABSTRACT Biofilm formation is an important characteristic of many Staphylococcus aureus infections because it limits the efficacy of host defenses and conventional antibiotic therapy. There are many literature reports that describe small molecule inhibitors of S. aureus biofilm formation, but the lack of direct comparisons and differences in the methods used to assess inhibition make it impossible to assess the efficacy of these compounds relative to each other. To address this, we compared 19 compounds reported in the literature to be inhibitors of S. aureus biofilm formation. We used the methicillin-susceptible clinical osteomyelitis isolate UAMS-1 in a microtiter plate biofilm assay shown to maximize biofilm formation and provide results consistent with those observed in vivo. Under these conditions, telithromycin (Ketek) showed the greatest inhibitory activity, limiting biofilm formation to a degree comparable to a UAMS-1 sarA mutant at a concentration of 0.49 µM (0.40 µg/mL). Similar results were obtained for the methicillin-resistant USA300 strain LAC. Telithromycin is a bacteriostatic ketolide antibiotic that is not currently approved in the United States for use as an antibiotic due to its concerning safety profile. However, the concentration required to limit growth in vitro was higher than the concentration required to limit biofilm formation, suggesting that low-dose telithromycin might be useful to limit biofilm formation and increase the efficacy of other antibiotics in biofilm-associated infections. Irrespective of whether telithromycin is ultimately proven clinically useful in this regard, these results emphasize the need for widespread use of a standardized in vitro approach to evaluate prospective inhibitors. IMPORTANCE Because biofilm formation is such a problematic feature of Staphylococcus aureus infections, much effort has been put into identifying biofilm inhibitors. However, the results observed with these compounds are often reported in isolation, and the methods used to assess biofilm formation vary between labs, making it impossible to assess relative efficacy and prioritize among these putative inhibitors for further study. The studies we report address this issue by directly comparing putative biofilm inhibitors using a consistent in vitro assay. This assay was previously shown to maximize biofilm formation, and the results observed with this assay have been proven to be relevant in vivo. Of the 19 compounds compared using this method, many had no impact on biofilm formation under these conditions. Indeed, only one proved effective at limiting biofilm formation without also inhibiting growth.

ABSTRACTWe previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus.

Proximal tubule endocytosis is essential to produce protein-free urine as well as to regulate system-wide metabolic pathways, such as the activation of Vitamin D. We have determined that the proximal tubule expresses an endolysosomal membrane protein, protein spinster homolog1 (Spns1), which engenders a novel iron conductance that is indispensable during embryonic development. Conditional knockout of Spns1 with a novel Cre-LoxP construct specific to megalin-expressing cells led to the arrest of megalin receptor-mediated endocytosis as well as dextran pinocytosis in proximal tubules. The endocytic defect was accompanied by changes in megalin phosphorylation as well as enlargement of lysosomes, confirming previous findings in Drosophila and Zebrafish. The endocytic defect was also accompanied by iron overload in proximal tubules. Remarkably, iron levels regulated the Spns1 phenotypes because feeding an iron-deficient diet or mating Spns1 knockout with divalent metal transporter1 knockout rescued the phenotypes. Conversely, iron-loading wild-type mice reproduced the endocytic defect. These data demonstrate a reversible, negative feedback for apical endocytosis and raise the possibility that regulation of endocytosis, pinocytosis, megalin activation, and organellar size and function is nutrient-responsive. NEW & NOTEWORTHY: Spns1 mediates a novel iron conductance essential during embryogenesis. Spns1 knockout leads to endocytic and lysosomal defects, accompanied by iron overload in the kidney. Reversal of iron overload by restricting dietary iron or by concurrent knockout of the iron transporter, DMT1 rescued the endocytic and organellar defects and reverted markers of iron overload. These data suggest feedback between iron and proximal tubule endocytosis. [ABSTRACT FROM AUTHOR]

The BAFF-APRIL system is crucial for the pathogenesis of systemic lupus erythematosus (SLE) by promoting B cell survival, differentiation and the maintenance of humoral autoimmunity. Here, we investigated the relationship of BCMA expression on B cell subsets with its ligands BAFF and APRIL, together with soluble BCMA, and with clinical and serologic variables in a cohort of 100 SLE patients (86 under conventional and 14 under belimumab therapy) and 30 healthy controls (HCs) using multicolor flow cytometry and ELISA. We found that BCMA expression in SLE patients was significantly increased on all B cell subsets compared to HCs, with all examined components of the BAFF-APRIL system being upregulated. BCMA expression was significantly increased on switched and unswitched memory B cells compared to naïve B cells, both in HCs and SLE. BCMA expression on B cells correlated with plasmablast frequencies, serum anti-dsDNA antibodies and complement consumption, while soluble BCMA correlated with plasmablast frequency, highlighting its potential as a clinical biomarker. Belimumab treatment significantly reduced BCMA expression on most B cell subsets and soluble TACI and contributed to the inhibition of almost the entire BAFF-APRIL system and restoration of B cell homeostasis. These results provide insights into the complex dysregulation of the BAFF-APRIL system in SLE and highlight the therapeutic potential of targeting its components, particularly BCMA, in addition to its use as a biomarker for disease activity. [ABSTRACT FROM AUTHOR]

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Measurement(s) area of attack of enemy species on non-native tree • intensity of attack of enemy species on non-native tree Technology Type(s) species identification • visual observation method Factor Type(s) plant health • country • species Sample Characteristic - Organism tree Sample Characteristic - Environment forested area Sample Characteristic - Location Europe Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.14345921

Abstract We previously reported the development of an osteogenic bone filler scaffold consisting of degradable polyurethane, hydroxyapatite, and decellularized bovine bone particles. The current study was aimed at evaluating the use of this scaffold as a means of local antibiotic delivery to prevent infection in a bone defect contaminated with Staphylococcus aureus. We evaluated two scaffold formulations with the same component ratios but differing overall porosity and surface area. Studies with vancomycin, daptomycin, and gentamicin confirmed that antibiotic uptake was concentration dependent and that increased porosity correlated with increased uptake and prolonged antibiotic release. We also demonstrate that vancomycin can be passively loaded into either formulation in sufficient concentration to prevent infection in a rabbit model of a contaminated segmental bone defect. Moreover, even in those few cases in which complete eradication was not achieved, the number of viable bacteria in the bone was significantly reduced by treatment and there was no radiographic evidence of osteomyelitis. Radiographs and microcomputed tomography (µCT) analysis from the in vivo studies also suggested that the addition of vancomycin did not have any significant effect on the scaffold itself. These results demonstrate the potential utility of our bone regeneration scaffold for local antibiotic delivery to prevent infection in contaminated bone defects.

In 2023, the invasive black twig borer Xylosandrus compactus (Eichhoff, 1875) (Coleoptera: Scolytinae) was found for the first time in Switzerland. Outbreaks of this non-native species have been recorded during phytosanitary survey activities in four municipalities south of the Alps in the Canton of Ticino. Conducting genetic analyses using the mitochondrial COI barcode region, specimens found in Switzerland were identical with those from France, Italy, and Spain, suggesting a European source of invasion. So far, only ornamental plants in urban areas were found to be affected by attacks of X. compactus. However, because of the increasing proportion of broadleaved evergreen plant species, the availability of suitable host plants such as Laurus nobilis may also favour the spread of X. compactus into insubric forest systems. Three previously undescribed host plant species, Argyrocytisus battandieri, Mahonia aquifolium, and Wisteria sp., were identified. In addition, fungal isolations from adult beetles and breeding galleries revealed eight fungal species likely or potentially associated with X. compactus. These include Ambrosiella xylebori (Ascomycota: Ceratocystidaceae), the obligate ambrosia fungus of X. compactus, and a Thyridium species (Ascomycota: Thyridiaceae) that has been found as an associated fungus in previous studies. [ABSTRACT FROM AUTHOR]

Using DNA affinity chromatography we demonstrate that the S. aureus regulatory proteins MgrA, Rot, SarA, and SarS bind DNA baits derived from the promoter regions associated with the genes encoding aureolysin, ScpAB, SspABC, and SplA-F. Three of four baits also bound SarR and SarZ, the exception in both cases being the ScpAB-associated bait. Using the USA300, methicillin-resistant strain LAC and the USA200, methicillin-sensitive strain UAMS-1, we generated mutations in the genes encoding each of these proteins alone and in combination with sarA and examined the impact on protease production, the accumulation of high molecular weight proteins, and biofilm formation. These studies confirmed that multiple regulatory loci are involved in limiting protease production to a degree that impacts all of these phenotypes, but also demonstrate that sarA plays a predominant role in this regard. Using sarA mutants unable to produce individual proteases alone and in combination with each other, we also demonstrate that the increased production of aureolysin and ScpA is particularly important in defining the biofilm-deficient phenotype of LAC and UAMS-1 sarA mutants, while aureolysin alone plays a key role in defining the reduced accumulation of alpha toxin and overall cytotoxicity as assessed using both osteoblasts and osteoclasts.

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU–tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.